RESUMEN
Damaged proteins containing abnormal isoaspartyl (isoAsp) accumulate as seeds age and the abnormality is thought to undermine seed vigor. Protein-L-isoaspartyl methyltransferase (PIMT) is involved in isoAsp-containing protein repair. Two PIMT genes from rice (Oryza sativa L.), designated as OsPIMT1 and OsPIMT2, were isolated and investigated for their roles. The results indicated that OsPIMT2 was mainly present in green tissues, but OsPIMT1 largely accumulated in embryos. Confocal visualization of the transient expression of OsPIMTs showed that OsPIMT2 was localized in the chloroplast and nucleus, whereas OsPIMT1 was predominately found in the cytosol. Artificial aging results highlighted the sensitivity of the seeds of OsPIMT1 mutant line when subjected to accelerated aging. Overexpression of OsPIMT1 in transgenic seeds reduced the accumulation of isoAsp-containing protein in embryos, and increased embryo viability. The germination percentage of transgenic seeds overexpressing OsPIMT1 increased 9-15% compared to the WT seeds after 21-day of artificial aging, whereas seeds from the OsPIMT1 RNAi lines overaccumulated isoAsp in embryos and experienced rapid loss of seed germinability. Taken together, these data strongly indicated that OsPIMT1-related seed longevity improvement is probably due to the repair of detrimental isoAsp-containing proteins that over accumulate in embryos when subjected to accelerated aging.
Asunto(s)
Oryza/enzimología , Proteínas de Plantas/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Ácido Abscísico/biosíntesis , Secuencia de Aminoácidos , Genes de Plantas , Datos de Secuencia Molecular , Oryza/embriología , Oryza/genética , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Interferencia de ARN , Elementos Reguladores de la Transcripción , Semillas/enzimología , Semillas/genética , Homología de Secuencia de Aminoácido , Estrés Fisiológico , Fracciones Subcelulares/metabolismoRESUMEN
PURPOSE: Cortactin is commonly expressed in several human cancers, which may alter their invasive or metastatic properties. Eighty five kilodalton form (p85) and 80-kDa form (p80) of cortactin are two separate bands in SDS-PAGE representing different conformational states. The objective of this study was to investigate cortactin expression in colorectal cancer (CRC). EXPERIMENTAL DESIGN: Cortactin expression was studied in an eight paired laser capture microdissection (LCM) CRC tissues and matched non-cancerous epithelia by immunoblotting. The expression in 58 CRC and two cell lines, HCT8 and HCT116, was studied respectively by immunohistochemistry and confocal laser scanning immunofluorescence. RESULTS: Dominant expression of p85 was identified in LCM-procured CRC tissues compared with equal intensity of p85 and p80 forms in non-cancerous tissues, while the amount of total cortactin was approximate. Immunohistochemistry analysis demonstrated that cortactin located in the cytoplasm of tumor cells and adjacent non-cancerous cells, and its expression was negatively correlated with TNM staging and lymphatic invasion status. However, the invasion fronts in 3 of 58 primary tumors and 28 of 39 available lymph node metastases were intensively stained. Further, immunofluorescence analysis showed that cortactin was distributed in cytoplasm and enriched in the front of the extending lamellipodia at adhering side of cultured cancer cells. CONCLUSIONS: Our results demonstrated the dominant expression of p85 form of cortactin in CRC for the first time. The enrichment of cortactin in the invasion front of some tumor cells and in the extending lamellipodia of cultured cancer cells suggests that cortactin may help cancer cell movement.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Cortactina/análisis , Movimiento Celular , Neoplasias Colorrectales/patología , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Mucosa Intestinal/química , Terapia por Láser , Masculino , Microdisección/métodos , Persona de Mediana Edad , Estadificación de Neoplasias , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The immediate early proteins ICP0 and BICP0 from Herpes virus are promiscuous activators of both viral and cellular genes and play a critical role in virus life cycle. Here we report that ICP0 and BICP0 could induce NF-kappaB translocation from cytoplasm into nucleus and strongly activate NF-kappaB responsive genes specifically. This process was dependent on the RING domain of both proteins. In addition, ICP0 interacted specifically with IkappaBalpha and its activating effect was attenuated by Ubch5A(C85A) and MG132, but not by IkappaBalpha(S32A/S36A). Remarkably, IkappaBalpha was poly-ubiquitinated by both ICP0 and BICP0, in vitro and in vivo. These data indicate that ICP0 and BICP0, functioning as ubiquitin ligases, are bona fide activators of NF-kappaB signaling pathway. Our study identifies a new way ICP0 and BICP0 explore to regulate gene expression.
Asunto(s)
Proteínas I-kappa B/metabolismo , Proteínas Inmediatas-Precoces/fisiología , FN-kappa B/metabolismo , Transactivadores/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/fisiología , Animales , Bovinos , Línea Celular , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Ensayo de Cambio de Movilidad Electroforética , Regulación Viral de la Expresión Génica , Humanos , Quinasa I-kappa B , Proteínas I-kappa B/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Leupeptinas/farmacología , Mutación , Inhibidor NF-kappaB alfa , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/fisiología , Simplexvirus , Secuencias Repetidas Terminales/genética , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción ReIA , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transfección , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The immediate early protein ICP0 encoded by herpes simplex virus 1 (HSV-1) is believed to activate transcription and consequently productive infection. The precise mechanisms of ICP0-mediated transactivation are under intensive study. Here, we demonstrate that ICP0 can strongly activate AP-1 responsive genes specifically. This activation is inhibited by c-Jun (S73A), c-Jun (S63/73A), TAK1 (K63W), but not by p38 (AF), ERK1 (K71R), ERK2 (K52R) and TRAF6 (C85A/H87A). We further investigate the relevancy of ERK, JNK and p38 MAPK pathways using their respective inhibitors PD98059, SP600125 and SB202190. Only SP600125 significantly attenuates the AP-1 responsive gene activation by ICP0. Consistent with these, the JNK is remarkably activated in response to ICP0, and this JNK activation is shown to be significantly attenuated by TAK1 (K63W). It turns out that ICP0 interacts specifically with TAK1 and stimulates its kinase activity. These findings reveal a new molecular mechanism ICP0 explores to regulate gene expression.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Línea Celular , Línea Celular Tumoral , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Modelos Biológicos , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/fisiología , Ubiquitina-Proteína Ligasas , Regulación hacia Arriba/fisiologíaRESUMEN
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological processes, including adaptive immunity, innate immunity, and bone metabolism. Importantly, it is essential for activating NF-kappaB signaling pathway in response to interleukin-1 and Toll-like receptor ligands. Previously, we characterized TRAF6 to be a ubiquitin ligase. In combination with the ubiquitin conjugating enzyme complex Ubc13/Uev1A, TRAF6 could catalyze the formation on itself of unique Lys-63 linked polyubiquitin chain that positively regulated NF-kappaB signaling pathway. However, it remains unknown how this auto-ubiquitination process is regulated. In this study, we found that the coiled-coil domain of TRAF6 was essential for its auto-ubiquitination and activating NF-kappaB signaling pathway. This domain served not as the specific target where the polyubiquitin chain was linked, but as a specific bridge to recruit Ubc13/Uev1A.